Facile Assembly of Soft Nanoarchitectures and Co-Loading of Hydrophilic and Hydrophobic Molecules via Flash Nanoprecipitation

ABSTRACT

Described herein are flash nanoprecipitation methods capable of encapsulating hydrophobic molecules, hydrophilic molecules, bioactive protein therapeutics, or other target molecules in amphiphilic copolymer nanocarriers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/656,905, filed Jul. 21, 2017, which claims priority to U.S.Provisional Application No. 62/365,849, filed Jul. 22, 2016, each ofwhich is incorporated herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CBET1453576awarded by the National Science Foundation. The government has certainrights in the invention.

BACKGROUND

Nanocarriers present a versatile method of controlled delivery forbioactive molecules that may otherwise be too hydrophobic or susceptibleto degradation for therapeutic applications. A key parameter ofnanocarrier design is the nanoarchitecture, which strongly influences invivo transport, biodistribution, and cellular uptake. The ability totailor nanocarrier architecture has resulted in numerous advancements intargeted delivery, for example, providing enhanced circulation time,membrane permeation and the simultaneous loading of multiple moleculesthat differ in water solubility. The self-assembly of block-copolymersallows the formation of diverse soft nanoarchitectures, but presentsseveral engineering challenges, namely: loading efficiency, scalability,repeatability and ease of fabrication, among others. Flashnanoprecipitation (FNP) is a fabrication technique capable of addressingthe majority of these issues, but has so far only been applied for theformation of solid-core nanoparticles and their loading with hydrophobicdrugs.

SUMMARY OF THE INVENTION

In a first aspect, provided herein is a method for preparingnanocarriers by flash precipitation comprising the steps of: (i)providing an organic phase solution comprising an amphiphilic copolymerand a process solvent, wherein the amphiphilic copolymer has a glasstransition temperature below 0° C., (ii) providing an aqueous phasesolution comprising an aqueous solvent, (iii) mixing the organic phasesolution and the aqueous phase solution to form a mixture, and (iv)introducing the mixture into a reservoir to cause precipitation of theamphiphilic copolymer as a nanocarrier. In some embodiments, the processsolvent is selected from the group consisting of tetrahydrofuran (THF),dimethylformamide (DMF), and dimethyl sulfoxide (DMSO). In someembodiments, the aqueous solvent is water.

In some embodiments, the amphiphilic copolymer has a glass transitiontemperature between about −40° C. and about 0° C. In some embodiments,the amphiphilic copolymer is poly(ethylene glycol)-bl-poly(propylenesulfide) (PEG-bl-PPS). In one embodiment, the copolymer isPEG₁₇-bl-PPS₃₀-Thiol.

In some embodiments, the organic phase solution additionally comprisesone or more target molecules. In some embodiments, the aqueous phasesolution additionally comprises a target molecule. In some embodiments,the target molecule is selected from the group consisting of a DNAmolecule, an RNA molecule, a plasmid, a peptide, a protein, andcombinations thereof.

In some embodiments, the reservoir comprises an aqueous nonsolvent. Insome embodiments, the reservoir comprises a target molecule.

In some embodiments, the mixing is by impingement. In some embodiments,the mixing comprises at least 2 impingements.

In a second aspect, provided herein is a method for preparingnanocarriers by flash precipitation comprising the steps of: (i)providing an organic phase solution comprising an amphiphilic copolymerand a process solvent, wherein the amphiphilic copolymer has a glasstransition temperature below 0° C., (ii) providing an aqueous phasesolution comprising an aqueous solvent and an target molecule, (iii)mixing the organic phase solution and the aqueous phase solution to forma mixture, and (iv) introducing the mixture into a reservoir to causeprecipitation of the amphiphilic copolymer as a nanocarrier loaded withthe target molecule.

In some embodiments, the target molecule is selected from the groupconsisting of a DNA molecule, an RNA molecule, a plasmid, a peptide, aprotein, and combinations thereof.

In some embodiments, the process solvent is selected from the groupconsisting of tetrahydrofuran (THF), dimethylformamide (DMF), anddimethyl sulfoxide (DMSO).

In some embodiments of the second aspect, the aqueous solvent is water.In some embodiments, the amphiphilic copolymer has a glass transitiontemperature between about −40° C. and about 0° C. In some embodiments,the amphiphilic copolymer is poly(ethylene glycol)-bl-poly(propylenesulfide) (PEG-bl-PPS). In one embodiment, the copolymer isPEG₁₇-bl-PPS₃₀-Thiol.

In some embodiments of the second aspect, the organic phase solutionadditionally comprises an target molecule.

In a third aspect, provided herein is a method for preparingnanocarriers by flash precipitation comprising the steps of: (i)providing an organic phase solution comprising an amphiphilic copolymerand a process solvent, wherein the amphiphilic copolymer has a glasstransition temperature below 0° C., (ii) providing an aqueous phasesolution comprising an aqueous solvent and an target molecule, (iii)mixing the organic phase solution and the first aqueous phase solutionto form a mixture, (iii) portioning the mixture into a first portion anda second portion, (iv) mixing the first portion and the second portionto form a second mixture, and (v) introducing the second mixture into areservoir to cause precipitation of the amphiphilic copolymer as ananocarrier loaded with the target molecule. In some embodiments, steps(iii) and (iv) are repeated at least one time.

In some embodiments of the third aspect, the organic phase solutionadditionally comprises a second target molecule.

In some embodiments, the mixing is by impingement.

In a fourth aspect, provided herein is a nanocarrier made by any of themethods described herein.

BRIEF DESCRIPTION OF DRAWINGS

The patent or patent application file contains at least one drawing incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1C show an overview of polymersome formation by flashnanoprecipitation (FNP). FIG. 1A shows a schematic of the CIJ mixer.FIG. 1B shows the structure of the diblock copolymer poly(ethyleneglycol)-block-poly(propylene sulfide), and the weight fraction (fPEG)dependent nanostructure known to form using the thin film hydrationmethod. FIG. 1C shows a representative cryoTEM image of polymersomesformed by FNP, scale bar+300 nm. Insert is a size distribution ofpolymersomes measured by nanoparticle tracking analysis (NTA), n=6.Standard deviation is represented by the dotted lines.

FIG. 2A-2G show fabrication of monodisperse polymersomes via flashnanoprecipitation. FIG. 2A shows DLS mean diameter of polymersomesformed after multiple inpingements (1×-5×), or formed by thin film (TF)or solvent dispersion (SD) with (E) or without (NE) extrusion. Errorbars are standard error, n=5. FIG. 2B shows DLS distribution of 5×impinged polymersomes the day of formation or after four days of storageat room temperature. Error bars are standard error, n=3. FIGS. 2C-2Gshow CryoTEM images of polymersomes formed after multiple impingements(1×-5×, respectively) with inserts of DLS size distributions. X- andy-axes correspond to that of FIG. 2B.

FIGS. 3A-3G show the relationship between PEG weight fraction andmorphology. FIG. 3A shows diameter of nanostructures formed via FNP fromPEG-bl-PPS copolymers of varying block lengths. Error bars represent thestandard deviation of the nanostructure populations (PDI×Mean Diameter).Dotted area represents polymersomes-forming samples. Arrows point outsamples of note. †Samples formed using DMF as the organic solvent,rather than THF. ‡Sample formed using water instead of 1×PBS. FIGS.3B-3G show weight fractions of PEG responsible for forming specificnanostructures via flash nanoprecipitation, paired with cartoon andrepresentative cryoTEM images. All scale bars=100 nm, with the exceptionof scale bars within FIGS. 3B and 3E, which are 300 nm. Sample number islisted in the upper corner of each cryoTEM image, and the number ofimpingements used is listed for each morphology. See Table 1 for detailsof the copolymers and FIGS. 10A-10E for low magnification images.

FIGS. 4A-4D show loading of polymersomes with small molecules andmacromolecules. (A) Loading efficiency of small molecules andmacromolecules. (B) Live-cell confocal microscopy image of polymersomeuptake and delivery of GFP in a bone marrow-derived dendritic cell.Polymersomes were loaded with the hydrophobic ethyl eosin (red) andhydrophilic GFP (green). Cells were additionally stained with SYTO 61(yellow) and lysotracker (blue). Scale bar=5 microns. (C) Graphicalrepresentation of experimental setup. Alkaline phosphatase (AP) isrepresented by circles, BCIP by triangles, and NBT by squares. Theproduct of the enzymatic reaction, formazan, absorbs strongly at 620 nmand is represented by a star. (D) Time-course of enzyme activity assay.Y-axis represents fold increase over original absorbance reading. Errorbars represent standard deviation, n=4. Statistical significancedetermined by 2-way ANOVA, *p<0.05 and ***p<0.001.

FIGS. 5A-5D show in vivo delivery of theranostic rapamycin/DiD-loadedpolymersomes formed by flash nanoprecipitation. FIG. 5A shows thepercentage of CD8+ T cells (CD45+CD3+CD4-CD8+) and CD4+ T cells(CD45+CD3+CD4+CD8-) within the total T cell population (CD45+CD3+) andpercentage of CD8+ DCs (CD11c+I-A/I-E+CD8+) within the total DC (CD11c+)population. Treatment groups were rapamycin polymersomes (R-PS), freerapamycin, blank polymersomes, and vehicle (PBS). FIG. 5B shows T cellsubpopulations as a percent of total T cell population for all fourtreatment groups. FIG. 5C shows T cells in the spleen and lymph nodes,as a percentage of CD45+ cells. FIG. 5D shows median fluorescenceintensity of the polymersome channel for selected cell populations inthe spleen and lymph nodes of mice administered rapamycin/DiD-loadedpolymersomes. N=3, statistical significance determined by Tukey'smultiple comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

FIGS. 6A-6D show efficiency of loading via syringe or reservoir. (A)Loading efficiency of fluorescently-labeled 10 kDa dextrans (CB=cascadeblue, F=fluorescein). Error bars represent standard deviation, n=6. (B)Loading efficiency of GFP when loaded via either syringe or reservoir.Error bars represent standard deviation, n=3. (C) Fluorescence ofsepharose CL-6B column separated GFP-loaded polymersomes loaded viasyringe or reservoir, 200 μL fractions. (D) Fluorescence of sepharoseCL-6B column separated GFP processed via FNP through the syringe orreservoir without PEG-bl-PPS copolymer, as a control. Included is atrace of blank polymersomes, for reference.

FIG. 7 shows DLS mean polydispersity (PdI) of polymersomes formed aftermultiple impingements (1×-5×), or formed by thin film (TF) or solventdispersion (SD) with (E) or without (NE) extrusion. Error bars arestandard error, n=5.

FIGS. 8A-8B show CryoTEM images of copolymers 5 and 9. (A) Micelles wereformed by copolymer 5. (B) Micelles and occasional polymersomes wereformed by copolymer 9. All scale bars represent 100 nm.

FIG. 9 shows emission spectra of calcein-DiI dual loaded polymersomes.Calcein-DiI polymersome emission (490 nm excitation) is represented bythe solid black line. Calcein and DiI emission spectra are included inthe plot for reference.

FIGS. 10A-10E show low magnification cryoTEM images of nanostructuresformed by FNP. (A) Polymersomes, (B) filomicelles, (C) tubularpolymersomes, (D) bicontinuous nanospheres, and (E) multilamellarpolymersomes. Scale bar represents 500 nm, except for in (E), where itrepresents 1500 nm.

FIG. 11 shows live-cell confocal microscopy image of polymersome uptakeand delivery of GFP in a RAW 264.7 cell. Polymersomes were loaded withthe hydrophobic ethyl eosin (red) and hydrophilic GFP (green). Cellswere additionally stained with SYTO (yellow) and lysotracker (blue).Scale bar=5 microns.

FIGS. 12A-12H show percentages of total live cells in the lymph nodes(FIGS. 12A, 12C, 12E, 12G) and spleen (FIGS. 12B, 12D, 12F, 12H) thatare T cells, dendritic cells (DCs), plasmacytoid dendritic cellsmonocytes (Mo), B cells, granulocytes, macrophages (Mφ), andneutrophils. † indicates rapamycin polymersome treated populations thatwere significantly altered compared to blank polymersomes. * indicatesfree rapamycin treated populations that were significantly alteredcompared to vehicle.

FIG. 13 depicts gating strategy for immune cells isolated from mousespleen to identify natural killer (NK) cells, B cells, CD4+ T cells,CD8+ T cells, ‘double negative’ (DN) T cells, and regulatory T cells (TReg). All plots shown are representative gating strategies for cells, inthis case from the spleen of a vehicle treated mouse.

FIG. 14 shows percentage of DN T cells within the CD45+ cell populationin the lymph nodes and spleen. N=3.

FIG. 15 shows cryoTEM images of loading of DNA into nanocarrierarchitectures. The rope-like structures within the vesicles shows loadedDNA.

FIG. 16 shows a comparison of the knockdown efficiency of siRNA-loadedpolymersomes compared to a commercially available gene deliveryplatform. LyoVec is the commercial gene delivery platform and cationicPS are polymersomes composed of copolymers that were end-functionalizedwith amines to give them a positive charge for better association withnucleic acids. Loaded siRNA was specific for glyceraldehyde 3-phosphatedehydrogenase (GSPDH).

INCORPORATION BY REFERENCE

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though set forth in their entirety in the presentapplication.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are flash nanoprecipitation preparation methods for thepreparation of nanocarriers. The methods described herein assemble andload nanocarriers with therapeutic molecules by flash nanoprecipitation.

As used herein, “flash nanoprecipitation” (FNP) refers to a process inwhich a block copolymer is assembled into a nanocarrier architecture.FNP is also used to load the nanocarrier with a target molecule such asa therapeutic or diagnostic molecule. FNP methods of the presentinvention employ multi-stream mixers in which an organic solution and ablock copolymer dissolved in a suitable solvent are impinged upon anaqueous solution under turbulent conditions and subsequently introducedinto an aqueous reservoir (FIG. 1A). The supersaturated conditionsgenerated by the turbulent mixing induces precipitation of the blockcopolymer for stabilization of monodisperse nanoparticles which may beloaded with one or more target molecules. Mixing occurs over millisecondtimescales and is followed by transfer to a reservoir comprising asecond aqueous solution to strip away solvent still associating with theaggregated block copolymer. Similar FNP methods are known in the art(U.S. Pat. No. 8,137,699 and U.S. Patent Publication No. 2012/0171254,each of which is incorporated herein in its entirety), however themethods of embodiments of the present invention offer at least theadvantages of being capable of loading hydrophilic target molecules,such as, but not limited to, large hydrophilic macromolecules such asRNA, DNA and proteins, and creating more advanced nanocarrierarchitectures with high loading efficiency.

Nanocarriers can be produced from amphiphilic copolymers that aredissolved in a process solvent. After the amphiphilic copolymers aredissolved in the process solvent, thereby forming an organic phasesolution, the solution is rapidly mixed with a first aqueous solutionand nanocarriers are flash precipitated following introduction of themixture into a reservoir comprising a second aqueous solution. Thismixing can be achieved through various methods during which the mixingvelocity, number of impingements, temperature and reservoir volume arecontrolled. In addition, a target molecule can be added with theamphiphilic copolymer in the process solvent prior to mixing, or atarget molecule can be added to the first aqueous solution prior tomixing. It is also envisioned that one or more target molecules can beadded to both the process solvent and the aqueous solution resulting inthe loading of multiple target molecules into the nanocarrierarchitecture.

Nanocarriers formed by the methods of the present invention arecharacterized by complex or vesicular nanoarchitectures capable ofencapsulating or comprising as part of the nanocarrier a targetmolecule. Nanoarchitectures formed by the methods of embodiments of thepresent invention are bicontinuous and may be characterized as, forexample, nanospheres, filomicelles, cubisomes, vesicles, tubules, nestedvesicles, filaments, and vesicular, multilamellar and tubularpolymersomes. It is envisioned that any soft nanoarchitecture with aninternal chamber capable of encapsulating a target molecule may beformed by the methods of embodiments of the present invention. One ofskill in the art will appreciate that changes in the amphiphiliccopolymer, the mixing velocity, number of impingements, temperature andreservoir volume will impact the nanoarchitecture of the nanocarrierproduced by the methods of the present invention.

Polymersomes are comprised of three separate topological regions: aninner aqueous cavity, a hydrophobic membrane, and an external surface,that together allow for simultaneous or individual transport of bothwater soluble/hydrophobic and lipophilic/hydrophobic target molecules.Polymersomes may be vesicular, multilamellar or tubular. Polymersomesformed by the methods of embodiments of the present invention have apolydispersity index (PDI) of between about 0.01 and about 0.99. In someembodiments, the PDI is between 0.20 and 0.64. In one embodiment, thePDI is less than 0.15 and the polymersomes are monodisperse.

In the methods according to embodiments of the present invention, mixingof the organic phase solution and the aqueous solution occurs byimpingement mixing or turbulent mixing. Mixing may occur in the presentmethods any number of times (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10+,100+, 1000+, 10,000+ times or more) before the mixture is introduced tothe second aqueous phase solution. In embodiments of the method in whichthe mixing occurs two or more times, following the first mixing, themixture is separated into a first portion and a second portion and thetwo portions are impinged upon one another or mixed using the same meansas for the first mixing. The portioning and mixing steps may be repeateduntil the solutions have been mixed the desired number of times. In someembodiments, the solutions are mixed 2 times. In some embodiments, thesolutions are mixed 3 times. In some embodiments, the solutions aremixed 5 times. In some embodiments, the solutions are mixed 10 or moretimes. In some embodiments, the solutions are mixed 100 or more times.

Without being bound to any particular theory, it is believed that thenumber of impingements effects the nanoarchitecture of the nanocarrierproduced by the methods of the present invention. Multiple impingementshave shown to both decrease the mean polymersome diameter and lower thePDI to levels equivalent to those achievable by extrusion methods forpolymersome preparation. In some embodiments, the solutions are impingedabout 2 to 3 times and form tubule nanoarchitectures. In someembodiments, the solutions are mixed about 3 to 4 times and form nestedvesicle nanoarchitectures. In some embodiments, the solutions are mixedabout 4 or more times and form monodisperse polymersomes. In someembodiments, the solutions are mixed a sufficient number of times suchthat the resulting polymersomes are monodisperse. It is envisioned thatthe number of impingements may vary depending on the modification ofother variables.

Any impingement mixer or turbulent known in the art can be used in themethods of the present invention. It is minimally required that 2liquids are mixed under turbulent conditions, such as a mixer in which 2or more liquids flow to meet at a single point. A suitable mixer for usein the methods of the present invention may include one or more inletsin which the two solutions are introduced into the mixing vessel throughindependent inlet tubes. A suitable mixer may also include temperaturecontrolling elements to adjust or maintain the mixing at a suitabletemperature. A suitable temperature will be a temperature at which allcomponents are stable, such as, for example, a temperate at which anyincluded protein will remain folded and will not denature. In someembodiments the temperature is between 0° C. and 40° C. In someembodiments the temperature is between 4° C. and 37° C. For volatileorganic solvents the temperature range is below their boiling point. Itis envisioned that higher temperatures may be used with polymers. Anymixer capable of providing a sufficient mixing velocity with controlledintroduction of the organic phase solution and the first aqueous phasesolution could facilitate flash precipitation under the methods of thepresent invention. Examples of suitable mixers include, but are notlimited to, confined impingement jets mixers, impinging get mixers, T-jet mixers, opposed jet mixers, micromixers, and the like.

A sufficient mixing velocity or flow rate is considered to be a velocityat which turbulent mixing is achieved. Variables including fluiddensity, channel length and fluid viscosity will change based on thesolvent, polymer, mixer, and any target molecules present and willchange the mixing velocity necessary to achieve turbulent mixing. Insome embodiments, turbulent mixing will be achieved at a Reynold (RE)number greater than 4000 based on the following equation. In someembodiments, turbulent mixing at an RE greater than 4000 is ideal, buttransitional flows with an RE between 1000 to 4000 will be sufficient toinduce flash nanoprecipitation formation of nanocarriers.

${RE} = \frac{( {{fluid}\mspace{14mu}{density}} )( {{mixing}\mspace{14mu}{velocity}} )( {{channel}\mspace{14mu}{length}} )}{( {{fluid}\mspace{14mu}{viscocity}} )}$

A suitable temperature will be a temperature at which all components arestable, such as, for example, a temperate at which any included proteinwill remain folded and will not denature. In some embodiments thetemperature is between 0° C. and 40° C. In some embodiments thetemperature is between 4° C. and 37° C.

The reservoir volume used in embodiments of the methods of the presentinvention is sufficiently high such that the process solvent is rapidlystripped away from the amphiphilic copolymer causing precipitation andformation of nanocarriers. In some embodiments of the invention thereservoir-to-process solvent volume ratio is at least about 5:1 (i.e.,5:1, 6:1, 7:1, 8:1, 10:1, 12:1, 15:1, 18:1, 20:1, 25:1 or about 30:1).In some embodiments, the reservoir-to-process solvent volume ratio isgreater than 6:1. In some embodiments reservoir-to-process solventvolume ratio is between about 6:1 and about 20:1. In one embodiment, thereservoir-to process solvent volume ratio is less than 20:1, preferablyless than 10:1. In one embodiment, the reservoir-to-process solventvolume ratio is 6:1. The reservoir comprises an aqueous nonsolventsolution in which the polymer process solvent is miscible, but in whichthe hydrophobic blocks of the copolymer are insoluble. In someembodiments of the invention, the reservoir comprises a target moleculeto be loaded upon flash precipitation of the nanocarrier. Without beingbound by any particular theory, it is believed that upon introduction ofthe process solvent into the aqueous solution of the reservoir, theprocess solvent will disperse as it is miscible with water, but theinsoluble hydrophobic blocks of the copolymer will aggregate. Theaggregation of the hydrophobic blocks of the copolymer will becontrolled to some extent by the presence of the hydrophilic blocks ofthe copolymer which are soluble in the aqueous nonsolvent solution.

As used herein, the term “organic phase solution,” refers collectivelyto the solution comprising the process solvent, the amphiphiliccopolymer, and optionally one or more target molecules. The processsolvent may be any water miscible organic solvent in which thehydrophobic block of the amphiphilic copolymer is soluble. The properprocess solvent will be selected based on the identity andcharacteristics of the amphiphilic copolymer selected. Water miscibleorganic solvents are known in the art and include, without limitation,tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide(DMSO), acetonitrile, methanol, 1,2-Butanediol, 1,3-Butanediol,1,3-Propanediol, 1,4-Butanediol, 1,4-Dioxane, 1,5-Pentanediol,1-Propanol, 2-Butoxyethanol, 2-Propanol, acetaldehyde, acetic acid,acetone, butyric acid, diethanolamine, diethylenetriamine,dimethoxyethane, dimethyl sulfoxide, dimethylformamide, ethanol,ethylamine, ethylene glycol, formic acid, furfuryl alcohol, glycerol,methyl diethanolamine, methyl isocyanide, propanoic acid, propyleneglycol, pyridine, tetrahydrofuran, triethylene, and glycol. The organicphase solution may optionally comprise one or more lipophilic orhydrophobic target molecules. In one embodiment, the process solvent isTHF. In one embodiment, the process solvent is DMSO. In one embodiment,the process solvent is DMF.

Amphiphilic copolymers are comprised of sub-units or monomers that havedifferent hydrophilic and hydrophobic characteristics. Typically, thesesub-units are present in groups of at least two, comprising a block of agiven character, such as a hydrophobic or hydrophilic block. Dependingon the method of synthesis, these blocks could be of all the samemonomer or contain different monomer units dispersed throughout theblock, but still yielding blocks of the copolymer with substantiallyhydrophilic and hydrophobic portions. These blocks can be arranged intoa series of two blocks (diblock) or three blocks (triblock), or more,forming the backbone of a block copolymer. In addition, the polymerchain may have chemical moieties covalently attached or grafted to thebackbone. Such polymers are graft polymers. Block units making up thecopolymer can occur in regular intervals or they can occur randomlymaking a random copolymer. In addition, grafted side chains can occur atregular intervals along the polymer backbone or randomly making arandomly grafted copolymer. The ratio of the hydrophobic to hydrophilicblocks of the copolymer will be selected such that the soluble andinsoluble components are balanced and suitable aggregation for thedesired architectures. Exemplary embodiments of various ratios are shownin FIG. 3A.

Suitable amphiphilic copolymers of the present invention are thosepolymers with a low glass transition temperature (Tg) hydrophobic block,typically below 0° C. or between about −70° C. and about 0° C. (i.e.,less than about 10° C., 0° C., −5° C., −10° C., −20° C., −25° C., −30°C., −40° C., −45° C., −50° C., −60° C. or −70° C. and greater than about−70° C., −60° C., −50° C., −45° C., −40° C., −30° C., −25° C., −20° C.,−10° C., or −5° C.). Polymers within this range will exhibit highmobility between polymer chains. Polymers which fit thesecharacteristics include, without limitation, poly(ethylene glycol)(PEG), poly(propylene sulfide) (PPS), poly(ethylene sulfide),polycaprolactone, poly(dimethylsiloxane) and polyethylene. Polymers mayalso include chemical modifications or end caps. Chemical modificationand end caps may include, but are not limited to, thiol, benzyl, pyridyldisulfide, phthalimide, vinyl sulfone, aldehyde, acrylate, maleimide,and n-hydroxysuccinimide groups. The chemical modification of thepolymer may add a charged residue to the polymer or may be used tootherwise functionalize the polymer. In some embodiments of the presentinvention, the polymer is poly(ethylene glycol)-bl-poly(propylenesulfide) (PEG-bl-PPS). In one embodiment, the polymer isPEG₁₇-bl-PPS₃₀-Thiol.

In some embodiments of the organic phase solution, the Hildebrandsolubility parameter (δ) of the hydrophobic portion of the amphiphiliccopolymer is matched to the solubility parameter of the water miscibleorganic solvent to increase the mobility of the polymer in solution. Theproper organic process solvent will be selected based on the identityand characteristics of the amphiphilic copolymer selected. In oneembodiment, the amphiphilic copolymer is PEG-bl-PPS (PPS δ=18.6MPa^(1/2)) and the organic solvent is THF (δ=18.6 MPa^(1/2)). In someembodiments of the organic phase solution, the solubility parameter isdissimilar between the copolymer and the organic solvent which lowerschain flexibility and produces slower kinetics for nanostructuretransitions. In one embodiment, the amphiphilic copolymer is PEG-bl-PPS(PPS δ=18.6 MPa^(1/2)) and the organic solvent is DMF (δ=24.8MPa^(1/2)).

As used herein, the term “aqueous phase solution” refers collectively tothe solution comprising an aqueous nonsolvent and optionally one or moretarget molecules. The aqueous solution can comprise an aqueousnonsolvent solution comprising pure water, a buffering agent, salt,colloid dispersant or inert molecule, or combinations thereof. Theaqueous phase solution may comprise one or more buffers, one or moresalts, and one or more supplemental additive agents, such as inertdiluents, solubilizing agents, emulsifiers, suspending agents,adjuvants, wetting agents, reducing agents, isotonic agents, colloidaldispersants and surfactants. In some embodiments, the aqueous nonsolventis phosphate-buffered saline (PBS). In some embodiments, the salt is akosmotropic salt. In some embodiments, the buffer is selected formcommon buffers used for biochemical reactions and cell culture,including phosphate buffer saline (PBS),(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES),tris(hydroxymethyl)aminomethane (Tris), citric acid, and3-(N-morpholino)propanesulfonic acid (MOPS). In some embodiments, thesalt is a kosmotropic salt. In some embodiments, the salt is selectedfrom the group consisting of, sodium chloride, ammonium acetate,potassium chloride, monopotassium phosphate, disodium phosphate, sodiumacetate, and zinc chloride.

The aqueous phase solution is formulated in a manner sufficient tomaintain the stability of any target agent suspended or dissolvedtherein. For example, it is envisioned that if the additive target agentis selected from the group consisting of a DNA molecule, an RNAmolecule, and a protein molecule, the aqueous solution will have aproper pH and salinity such that the target molecule will maintainproper folding and stability while in solution. In some embodiments, theaqueous phase solution will have a physiologically relevant pH andsalinity appropriate for loading biological macromolecules into thenanocarriers. In one embodiment, the aqueous phase solution comprisesbetween about 0 mM and 200 mM salt. In one embodiment the aqueous phasesolution comprises less than or equal to 150 mM salt. In one embodiment,the aqueous phase solution has a pH between about 2.0 and 12.0. In oneembodiment the aqueous phase solution has a pH between about 5.0 and9.0. In one embodiment the aqueous phase solution has a pH between about7.0 and 8.0.

As used herein, the term “aqueous nonsolvent” refers to the water orother aqueous solvent solution present in the aqueous phase solution orin the reservoir solution. The amphiphilic copolymer is not solvent inthe nonsolvent, and the nonsolvent acts to strip the water miscibleorganic solvent away from the amphiphilic copolymer during the processof flash nanoprecipitation.

In another aspect of the invention, nanocarriers are made and includeone or more target molecules. The one or more target molecules may beadded to the organic phase solution, the aqueous phase solution, thereservoir, or combinations thereof. In some embodiment, a targetmolecule is included with the amphiphilic copolymer in the organic phasesolution. In some embodiments, the target molecule is present in theaqueous phase solution. In some embodiments, a first target molecule isincluded in the organic phase solution and a second target molecule isincluded in the aqueous phase solution. In some embodiments, the targetmolecule is included in the reservoir. The target molecule is combinedwith the amphiphilic copolymer in a ratio of 1:4 to 10:1 by weight orcharge. In one embodiment, the target molecule is mixed with theamphiphilic copolymer in at least a 1:2 ratio by weight. Preferably thetarget molecules is present in the mixture after mixing at aconcentration of at least 0.1% by weight, but more preferably theconcentration of target molecule is at least 0.2% by weight. In someembodiments the target molecule is included at between 0.1% and 20% byweight, between 1% and 15% by weight or between 1.5% and 12% by weight.The temperature and the pressure of the organic phase solution, theaqueous phase solution or the mixture thereof can be altered to allowcomplete dissolution of both the amphiphilic copolymer and the targetmolecule while maintaining a liquid phase.

As used herein, the term “target molecules” refers to any molecule to beloaded into the nanocarriers according to embodiments of the presentinvention. The target molecule may be hydrophobic, hydrophilic,lipophilic or amphiphilic. The target molecule may include hydrophilicmacromolecules such as RNA, DNA, plasmids, peptides, antibodies,proteins, fluorophores, carbohydrates, small molecule drugs, watersoluble synthetic polymers and combinations thereof. Target moleculesalso include adhesive or targeting moieties such as cell specificantibodies which target the nanocarrier to a specific cell type ortarget of interest. Examples of other target molecules that may be addedto nanoparticles by this process can be selected from, but are notlimited to, the known classes of drugs including immunosuppressiveagents such as cyclosporins (cyclosporin A), immunoactive agents,analgesics, anti-inflammatory agents, anthelmintics, anti-arrhythmicagents, antibiotics (including penicillins), anticoagulants,antidepressants, antidiabetic agents, antiepileptics, antihistamines,antihypertensive agents, antimuscarinic agents, antimycobacterialagents, antineoplastic agents, immunosuppressants, antithyroid agents,antiviral agents, anxiolytic sedatives (hypnotics and neuroleptics),astringents, beta-adrenoceptor blocking agents, blood products andsubstitutes, cardiac inotropic agents, contrast media, corticosteroids,cough suppressants (expectorants and mucolytics), diagnostic agents,diagnostic imaging agents, diuretics, dopaminergics (antiparkinsonianagents), haemostatics, immunological agents, lipid regulating agents,muscle relaxants, parasympathomimetics, parathyroid calcitonin andbiphosphonates, prostaglandins, radio-pharmaceuticals, sex hormones(including steroids), anti-allergic agents, stimulants and anoretics,sympathomimetics, thyroid agents, vasodilators, xanthines,anti-oxidants, preservatives, vitamins, nutrients, adjuvants, antigents,MRI contrast agents, metal (i.e., gold, iron oxide, and the like),nanomaterials (i.e., quantum dots, micelles), temperature sensitivepolymers (i.e., Poly(N-isopropylacrylamide)), polymer-drug conjugates,and biologics (referring collectively to any carbohydrate, protein,polypeptide, nucleic acid, combinations thereof and the like). Targetmolecules may also include combinations of, complexes of, mixtures of orother associations of any of the target molecules listed.

In some embodiments, the methods described herein support simultaneousloading of both hydrophobic and hydrophilic target molecules. Withoutbeing bound by any particular theory, it is envisioned that hydrophobictarget molecules may be loaded into the polymersome or nanocarriermembrane while hydrophilic target molecules may be loaded into aqueouslumen formed with in the polymersome or nanocarrier. It is alsoenvisioned that the loaded molecules, in particular loaded hydrophilicbiological macromolecules, such as DNA, RNA, and proteins, remain activefollowing lysis from the nanocarrier or polymersome formed by themethods described herein.

The loading efficiency of the target molecule in the nanocarriers by themethods of the present invention is measured as the ratio of targetmolecule encapsulated within the nanocarrier to the total amount oftarget molecule available for loading in the initial solution. Theloading efficiency is typically greater than about 40% for both proteinsand hydrophobic molecules (i.e., about 35%, about 40%, about 45%, about50%, about 60%, about 70%, about 80%, about 85%, about 90%, or anypercentages in between). In some embodiments of the invention, theloading efficiency is greater than at least 45%.

One or more supplemental additives can be added to the organic phasesolution or aqueous phase solution or to a stream of nanoparticles afterformation by flash precipitation to tailor the resultant properties ofthe nanoparticles or for use in a particular indication. Examples ofsupplemental additives include inert diluents, solubilizing agents,emulsifiers, suspending agents, adjuvants, wetting agents, reducingagents, sweetening, flavoring, and perfuming agents, isotonic agents,colloidal dispersants and surfactants such as, but not limited to, acharged phospholipid such as dimyristoyl phophatidyl glycerol; alginicacid, alignates, acacia, gum acacia, 1,3 butyleneglycol, benzalkoniumchloride, collodial silicon dioxide, cetostearyl alcohol, cetomacrogolemulsifying wax, casein, calcium stearate, cetyl pyridiniumn chloride,cetyl alcohol, cholesterol, calcium carbonate, Crodestas F-110®, whichis a mixture of sucrose stearate and sucrose distearate (of Croda Inc.),clays, kaolin and bentonite, derivatives of cellulose and their saltssuch as, but not limited to, hydroxypropyl methylcellulose (HMPC),carboxymethylcellulose sodium, carboxymethylcellulose and its salts,hydroxypropyl celluloses, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,noncrystalline cellulose; dicalcium phosphate, dodecyl trimethylaminonium bromide, dextran, dialkylesters of sodium sulfosuccinic (e.g.Aerosol OT® of American Cyanamid), gelatin, glycerol, glycerolmonostearate, glucose, p-isononylphenoxypolt-(glycidol), also known asOlin 10-G® or surfactant 10-G® (of Olin Chemicals, Stamford, Conn.);glucamides such as octanoyl-N-methylglucamide,decanoyl-N-methylglucamide; heptanoyl-N-methylglucamide, lactose,lecithin(phosphatides), maltosides such as n-dodecyl β-D-maltoside;mannitol, magnesium stearate, magnesium aluminum silicate, oils such ascotton seed oil, corn germ oil, olive oil, castor oil, and sesame oil;paraffin, potato starch, polyethylene glycols (e.g., the Carbowaxs 3350®and 1450®, and Carbopol 9340® of Union Carbide), polyoxyethylene alkylethers (e.g. macrogol ethers such as cetomacrogol 1000), polyoxyethylenesorbitan fatty acid esters (e.g. the commercially available Tweens® ofICI specialty chemicals), polyoxyethylene castor oil derivatives,polyoxyethylene sterates, polyvinylalcohol (PVA), polyvinylpyrrolidone(PVP), phosphates, 4(1,1,3,3-tetramethylbutyl) phenol polymer withethylene oxide and formaldehyde, (also known astyloxapol, superione, andtriton), all poloxamers and polaxamines (e.g., Pluronics F68LF®, F87®,F108® and tetronic 908® available from BASF Corporation Mount Olive,N.J.), pyranosides such as n-hexyl β-D-glucopyranoside, n-heptylβ-D-glucopyranoside; n-octyl-β-D-glucopyranoside, n-decylβ-D-glucopyranoside; n-decyl β-D-maltopyranoside; n-dodecylβ-D-glucopyranoside; quaternary ammonium compounds, silicic acid, sodiumcitrate, starches, sorbitan esters, sodium carbonate, solid polyethyleneglycols, sodium dodecyl sulfate, sodium lauryl sulfate (e.g., Duponol P®of DuPont corporation), steric acid, sucrose, tapioca starch, talc,thioglucosides such as n-heptyl β-D-thioglucoside, tragacanth,triethanolamine, Triton X-200® which is a alkyl aryl polyether sulfonate(of Rhom and Haas); and the like. The inert diluents, solubilizingagents, emulsifiers, adjuvants, wetting agents, isotonic agents,colloidal dispersants and surfactants are commercially available or canbe prepared by techniques know in the art. In some embodiments, theexcipients are selected from the group consisting of amphiphilicpolymers, urea, chaotropic salts, and kosmotropic salts. The propertiesof many of these and other pharmaceutical excipients suitable foraddition to the organic phase solution and aqueous phase solution beforeor after mixing are provided in Handbook of Pharmaceutical Excipients,7rd edition, editor Arthur H. Kibbe, 2000, American PharmaceuticalAssociation, London, the disclosure of which is hereby incorporated byreference in its entirety.

Colloidal dispersants or surfactants can be added to colloidal mixturessuch as a solution containing nanoparticles to prevent aggregation ofthe particles. In one embodiment of the invention, a colloidaldispersant is added to either the organic phase solution or aqueousphase solution prior to mixing. In one embodiment, the colloidaldispersant can include a gelatin, phospholipid or pluronic. Thedispersant is typically added in a ratio up to 2:1 with the one or moretarget molecule by weight. The use of a colloidal dispersant can preventnanoparticles from growing to a size that makes them unusable for theuse in the treatment of subjects.

In another embodiment of the invention, the target molecule is mixedwith the amphiphilic copolymer with a supplemental seeding molecule. Theinclusion of a supplemental seed molecule in the process solventfacilitates the creation of nanoparticles upon micromixing with thenonsolvent. Examples of a supplemental seed molecule include, but arenot limited to, a substantially insoluble solid particle, a salt, afunctional surface modifier, a protein, a sugar, a fatty acid, anorganic or inorganic pharmaceutical excipient, a pharmaceuticallyacceptable carrier, or a low molecular weight oligomer.

In one embodiment, a supplemental surfactant can be added to the organicphase solution or the aqueous phase solution. This process can beperformed with amphiphilic copolymer alone or with an organic phasesolution or aqueous phase solution containing one or more targetmolecule.

Preferably the nanocarrier compositions containing one or moreamphiphilic copolymers, with or without one or more target molecules,and with or without one or more supplemental additives which areproduced by a flash precipitation by the methods of the presentinvention have an average size less than 1060 nm and more preferablyless than about 700 nm, alternatively less than about 500, alternativelyless than about 400, alternatively less than about 200, alternativelyless than about 100, alternatively less than about 40 nm. In someembodiments the size is between 80-150 nm. For filamentous nanocarrierarchitectures, the average diameter is between 5 to 100 nm (i.e.,between 10-90 nm, between 15-80 nm, and between 20-70 nm) with lengthsof a micron or greater. The average size is on a weight basis and ismeasured by light scattering, microscopy, or other appropriate methods.

The nanocarriers produced by the flash precipitation process ofembodiments of the present invention can be post processed to yield asterile aqueous or non-aqueous solution or dispersion or could beisolated, such as via lylophilization and autoclaving, to yield asterile powder for reconstitution into sterile injectable solutions ordispersions. The nanoparticles can be combined with other acceptablecompounds for parenteral injection such as but not limited to one ormore of the following: water, ethanol, propyleneglycol,polyethyleneglycol, glycerol, vegetable oils, and ethyl oleate.Supplemental additives suitable for parenteral injection can also beused to tailor the composition to that suitable for a specific purpose.

In one embodiment, the stream of nanocarriers produced via flashprecipitation, is distilled to remove any toxic solvents and sterilefiltered using a 0.22 μm nominal pore size filter to yield a sterilesolution. In another embodiment, the organic phase solution and aqueousphase solution are sterilized prior to use and are flash precipitated ina sterile environment to produce a sterile formulation. In someembodiments, any post processing is also performed under sterileconditions.

The nanocarrier compositions produced by the methods described hereinvia flash precipitation may also contain supplemental additives usefulfor preserving, wetting, emulsifying, or dispensing the pharmaceuticalcomposition. Prevention of the growth of microorganisms can be ensuredby various antibacterial and antifungal agents, such as, but not limitedto, sorbic acid, parabens, phenol, chlorobutanol. It may be desirable toadd an antioxidant such as tocopherol or the like, or it may bedesirable to include isotonic agents, such as, but not limited to,sugars or sodium chloride.

In one embodiment, the nanocarriers formed via flash precipitation areisolated via distillation to remove toxic solvents such as THF, asupplemental additive is added, such as the cryoprotectant sucrose ortrehelose, and the material is lyophilized to obtain a powder.

In one embodiment, the nanocarrier compositions produced by the flashprecipitation methods of the present invention are formulated into asolid dosage form for oral administration such as capsules, tablets,pills, powders, and granules, or the like. In such solid dosage forms,the nanocarrier composition is admixed with one or more supplementaladditives falling into the following classes such as, but not limitedto, lubricants, buffering agents, wetting agents, adsorption, inertexcipients, binders, disintegrating agents, solution retarders,accelerators, adsorbents, or fillers or extenders or other componentscommonly used by those skilled in the art for production of solid dosageforms.

In an embodiment, a composition comprising nanocarriers of the presentinvention is a potent pharmaceutical containing one or more amphiphiliccopolymers, with or without one or more target molecules, and with orwithout one or more supplemental additives which are produced by a flashprecipitation method of the present invention. In some embodiments, thecomposition is a solid dosage form and due to its nanoparticulate sizeit is evenly dispersed throughout said solid dosage form admixture andyields a high content uniformity (quantity of material in each dose) notobtained if the drug was microparticulate.

In one embodiment, the nanocarrier compositions produced by the flashprecipitation methods of the present invention are formulated into apharmaceutically acceptable liquid dosage form for oral administrationsuch as a syrup, solution, emulsion, suspension, or elixir. In additionto the amphiphilic copolymer nanocarriers, the liquid dosage forms maycomprise inert diluents, solubilizing agents, oils, emulsifiers,adjuvants suspending agents, sweeteners, wetting agents, flavoringagents, perfuming agents or other compounds commonly used by thoseskilled in the art.

The nanocarrier compositions containing one or more amphiphiliccopolymers, with or without one or more target molecules, and with orwithout one or more supplemental additives which are produced by theflash precipitation methods of the present invention can be administeredto a subject, for example, humans and animals, via a number of meansincluding, but not limited to, orally, rectally, parenterally(intravenous, intramuscular, or subcutaneous), intracisternally,intravaginally, intraperitoneally, locally (in the form of powders,ointments or drops) or as a buccal or nasal spray.

The nanocarriers produced by the methods of the present invention canalso be used in immunotheranostic or theranostic application whichcombine immunotherapy and diagnostics. It is envisioned that targetmolecules used for immunotherapy can be incorporated along with targetmolecules used for imagine and diagnostics into a single nanocarrier ora composition comprising nanocarriers for theranostic treatments.

An advantage of the methods of embodiments of the present invention isthe ability to encapsulate hydrophilic target molecules without therequirement of further processing. For example, the methods describedherein require no extrusion steps. As such, the methods of the presentinvention can be carried out over a short period of time (e.g. overminutes via FNP as opposed to days via thin film hydration followed byextrusion). In some embodiments, the nanocarriers are assembled in 5minutes or less.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

Example 1

The embodiment described here demonstrates flash nanoprecipitation andloading of nanocarrier structures of PEG-bl-PPS with a variety of targetmolecules including rapamycin, fluorescent dyes, dextran among others.Protocols for FNP employ multi-stream mixers in which an organicsolution of solubilized hydrophobic drug and an amphiphilic blockcopolymer dissolved in a water-miscible common solvent are impinged uponan aqueous solution briefly under turbulent conditions and subsequentlyintroduced into an aqueous reservoir (FIG. 1A). The supersaturatedconditions generated by the turbulent mixing induces precipitation andnucleation of the hydrophobic solute and coprecipitation of the blockcopolymer for stabilization of monodisperse nanoparticles withhydrophobic drug cores. Mixing occurs over millisecond timescales and isfollowed by transfer to a reservoir of aqueous nonsolvent to strip awaysolvent still associating with the aggregated drug and block copolymer.While micelles can form within nanoseconds, the combined entropicallyand enthalpically driven transition in aggregate morphology tofilomicelles, bilayer sheets and vesicles occurs over much longertimescales. The glass transition (Tg) of the amphiphile's hydrophobicblock influences chain flexibility and as a result the timescale ofaggregate shape transformations, which can range from hours for glassyhigh Tg polymers to milliseconds for low Tg polymers. Thus the rapidmixing followed by an immediate increase in water content within thereservoir during FNP effectively minimizes the chain mobility of thehydrophobic copolymer blocks to stably lock the molecular orientation ofthe assembly, and this is particularly effective for high Tg polymerslike polystyrene. It was determined that the organic solvent must beremoved from the assembly quickly to prevent nanoparticle instabilityand ripening, which can be achieved via flash solvent evaporation orusing aqueous reservoirs with large volumes to decrease the solventconcentration. While recent focus has been placed on this process ofcompetitive aggregation for the scalable loading of nanoparticles withhydrophobic drugs, FNP was originally applied to achieve rapid changesin solvent quality for homogenous precipitation and self-assembly ofblock copolymers to investigate the mechanism and kinetics ofmicellization. However, FNP has not been shown to produce more complexself-assembled soft nanoarchitectures such as polymer vesicles (i.e.polymersomes) that would be capable of loading hydrophilic therapeuticsuntil the present Example.

Structurally analogous to liposomes, polymersomes possess enhancedphysical and chemical stability and have emerged as versatile drugdelivery vehicles. Polymersomes are comprised of three separatetopological regions: an inner aqueous cavity, a hydrophobic membrane andan external surface that together allow simultaneous transport of bothwater soluble and lipophilic payloads as well as incorporation ofadhesive and targeting moieties. A key advantage of the inner lumen isthe ability to encapsulate and protect sensitive biologics, such asenzymes and nucleic acids. The size and shape of these nanocarriersimpacts their biodistribution, systemic clearance, cellularinternalization, optical properties and overall therapeutic potential.Aggregate morphology can be specified by synthesizing block copolymerswith a particular hydrophilic weight fraction (typically >45% forpolymersomes), kinetically trapping metastable intermediate structuresduring self-assembly or modulating the vesicles after formation oftenusing shear forces or osmotic pressure gradients. These methods haveresulted in a wide range of nanostructures with unique properties andapplications, including tubule polymersomes, multilamellar nestedvesicles and bicontinuous nanostructures. High throughput synthesis ofthese structures remains a challenge, as most of these morphologies canrequire days for formation and represent only a small fraction of theassembled aggregate population. The most commonly used methods ofpolymersome formation from di- or tri-block polymers are diversevariations of thin film hydration, solvent dispersion and microfluidics.Of these methods, thin film hydration and microfluidics have proven tobe most amenable to the loading of protein biologics, as they avoidextensive exposure of payloads to organic solvents that can disruptprotein conformation and activity. Unfortunately, thin film hydrationcannot control for vesicle size and requires additional processingsteps, primarily by extrusion through nanoporous membranes. The currentmethods do not require an extrusion step providing a distinct advantageover prior methods. Microfluidics is low throughput (μL/min) andfabricates primarily microscale vesicles due to restrictions on channeldimensions. Thus although numerous methods have been developed forpolymersome formation, a need still exists for facile, rapid, highthroughput methods capable of both specifying nanostructure morphologyand loading diverse bioactive payloads.

Poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) is ahighly versatile self-assembling block copolymer that can be engineeredto stably form polymersomes and a variety of other nanoarchitectures.This ability is in part due to the low Tg of PPS (227 K), which permitshigh chain flexibility at room temperature and rapid transitions betweenmetastable aggregate morphologies. Depending on the molecular weightratio of the hydrophilic PEG and hydrophobic PPS blocks as well as themethod of assembly, PEG-bl-PPS can assemble into micelles, vesicles andfilomicelles. Even at relatively low MW, PEG-bl-PPS aggregates arehighly stable in dilute aqueous solutions as evidenced by anoctanol/water partition coefficient (log P) of 13.07 and a criticalmicelle concentration of 3×10⁻⁶ M. This stability has allowed PEG-bl-PPSnanocarriers to support the delivery of a variety of therapeuticmolecules and fluorophores both in vitro and in vivo. Furthermore, theoxidation-sensitivity of PPS provides a means for controlled degradationand payload release via photo-oxidation or cell-derived ROS as well assystemic clearance of the block copolymer through the kidneys. Smallscale assembly and loading of PEG-bl-PPS nanocarriers have been achievedvia thin film hydration, direct hydration, solvent extraction, andfreeze thaw-cycling, which, in the case of polymersomes, subsequentlyrequires extensive extrusion to form monodisperse nanocarriers.

The inventors discovered that under the standard conditions of use, FNPmay serve as a scalable and rapid method to assemble and load softnanoarchitectures in addition to micelles when coupled with a blockcopolymer containing a hydrophobe with a sufficiently low Tg, such asPEG-bl-PPS. The rapid millisecond timescale of PEG-bl-PPS transitionsbetween metastable intermediate morphologies more closely matches themixing time of FNP, which may allow kinetic entrapment of nanostructuresthat are otherwise impossible to observe outside of molecular dynamicsmodels. Here, the inventors demonstrate that by adjusting the aqueoussolution conditions, number of impingements and reservoir volume, FNPcan rapidly assemble PEG-bl-PPS block copolymers into bicontinuousnanospheres, filomicelles and vesicular, multilamellar and tubularpolymersomes. Many of these nanocarrier morphologies are difficult toachieve reproducibly and have not been previously reported forPEG-bl-PPS copolymer systems. The inventors additionally demonstratethat multiple impingements of polymersomes allowed further modulation ofthe obtained nanostructure and a rapid method of decreasingpolydispersity, which presents an alternative to more time-intensiveextrusion. Furthermore, this Example demonstrates that diversehydrophilic molecules, including proteins, could be stably encapsulatedwithin polymersomes via FNP and retain their bioactivity followingrelease. Co-loading of therapeutics with fluorescent dyes allowed rapidformation of theranostic polymersomes. As an immunotheranostic (i.e.,combining immunotherapy and diagnostics) application of thismethodology, we demonstrate enhanced immunosuppression of T cells by lowdosage subcutaneous (SC) injection of rapamycin loaded withinfluorescent polymersomes. Flow cytometric analysis allowed associationof the therapeutic effect with intracellular delivery to lymph noderesident and splenic macrophages. Our results enhance the versatility ofFNP and may provide a facile high throughput method to fabricatedrug-loaded polymersomes for biomedical applications.

Results and Discussion

Formation of Polymersomes Via Flash Nanoprecipitation

The rate of self-assembly strongly influences the nanoarchitecture ofblock copolymer aggregates, and for FNP, at a standard temperature(i.e., a temperature between 0° C. and 37° C. for nanocarriers that maycomprise biologics), is primarily determined by the polymerconcentration, steric stabilization of the hydrophilic block, similarityin solubility parameters of the common solvent and non-solvent for boththe hydrophilic and hydrophobic blocks, and the rate of removal of thesolvent upon addition to the non-solvent reservoir. Although many ofthese conditions have been optimized for specific block copolymerchemistries and drug loading, several standard practices have beenuniversally adopted for FNP protocols to achieve stable kineticallytrapped nanoparticles. These include the use of tetrahydrofuran (THF)and pure water as the solvent and nonsolvent, respectively, as well aslarge reservoir-to-solvent volume ratios. Decreasing the polymerconcentration as well as solvent selection have been employed to bothspecify nanoparticle size and to enhance payload complexation with theblock copolymer. The inventors therefore hypothesized that the use of adiblock copolymer with an appropriate hydrophilic to hydrophobic blockmolecular weight ratio and sufficiently low Tg may be induced to formpolymersomes under the millisecond mixing conditions of FNP. The Tg andHildebrand solubility parameter (δ) for PPS (δ=17.9 MPa^(1/2)) bothsuggested PEG-bl-PPS to be well suited for the kinetic entrapment ofmetastable aggregate morphologies using standard protocols for FNP in aconfined impingement jets (CIJ) mixer, which involves the impingement ofTHF (δ=18.6 MPa^(1/2)) with an aqueous solution at a flow rate of1.2×10⁻⁶ m³/s (FIG. 1A).

In order to investigate the potential for FNP to form polymersomes, theinventors used PEG₁₇-bl-PPS₃₀-Thiol, a polymer already demonstrated toform polymersomes via both thin film rehydration and solvent dispersion.This copolymer was dissolved in THF, impinged at 1.2×10⁻⁶ m³/s against1×PBS within a CIJ mixer and introduced into a 1×PBS aqueous reservoir(FIGS. 1A and 1B). The resulting assemblies were analyzed via cryoTEMand nanoparticle tracking analysis (NTA) to respectively assess theirmorphology and size, and were found to consist of polymersomes (FIG.1C). The use of a PBS salt solution served two purposes. First,vesicular nanoarchitectures maintain osmotic balance with downstreambiological fluids in which they will be applied, or risk rupture.Second, the inventors suspected that decreasing the PEG solubility andresulting rate of steric stabilization with a kosmotropic salt solutionwould allow increased chain mobility for vesicle assembly. To furtherenhance chain mobility, FNP was performed under solvent conditions whichnormally result in unstable Ostwald ripening and coalescence.Specifically, the volume ratio of THF/aqueous dilution within thereservoir was only 1:6, while ratios of 1:20 are often suggested inother implementations of FNP for rapid solvent extraction to kineticallytrap micellar nanostructures. Use of these conditions (decreased PEGsolubility and increased organic solvent concentration in the reservoir)likely provided a system of dynamic self-assembly that allowed continuedassembly of the PEG₁₇-bl-PPS₃₀-Thiol into polymersomes within theaqueous reservoir. This was later verified by the observed loading offluorescently tagged 10 kDa dextran from the reservoir into thepolymersome lumen (FIG. 6A). Switching the solvent to dimethylformamide(δ=24.8 MPa^(1/2)) resulted in the formation of micelles for copolymersthat were initially found to assemble vesicles when THF was used as thecommon solvent. This result may be due the lower similarity insolubility parameters between DMF and PPS relative to THF and PPS, whichwould lower chain flexibility and produce slower kinetics fornanostructure transitions. Slowing the transition kinetics through theuse of less similar common solvents is known to promote micelle insteadof vesicle formation for other block copolymer systems.

Multiple Impingements Reduce Polymersome Diameter and Polydispersity

Nanocarriers used in biomedical applications are typically expected tohave a low polydispersity for therapeutic reproducibility andconsistency in biodistribution and cellular targeting. The polymersomesformed by FNP (Table 1) demonstrated a polydispersity index (PDI) thatranged from 0.220 to 0.634, which is similar to polymersomes formed bysolvent dispersion and thin film rehydration that require subsequentextrusion through membranes with nanoscale porosity to achievemonodisperse vesicles (FIGS. 2A and 7). The extrusion process can betime-consuming, result in the loss of product, potentially reduceloading efficiency, and presents an opportunity for the introduction ofcontaminants and endotoxin. Shear flow has been demonstrated to be aviable and underexplored mechanism to influence the shape andpolydispersity of metastable aggregate states. Since vesicle uniformityhas been found to improve with increasing shear rate, we hypothesizedthat repeated impingement of polymersomes within the CIJ mixer underconditions promoting continued fluidity of the PPS membrane may decreasevesicle polydispersity without the need for subsequent extrusion.

TABLE 1 Relationship between PEG weight fraction (f_(PEG)) andmorphology. PEG PPS Com/Aqu # DoP DoP f_(PEG) End Capping Solv D (nm)PDI Morphology  1 17 75 0.119 Thiol THF/PBS 143.13 0.62 BN, M, P  2 1744 0.187 Benzyl THF/PBS 116.53 0.24 P  3 17 38 0.21 Thiol THF/PBS 115.290.63 P, MLP*, TP*  3‡ THF/Water N/A N/A FM  4 17 36 0.219 Thiol THF/PBS80.51 0.37 P, MLP*, TP*  4† DMF/PBS 41.53 0.31 M, P**  5 45 96 0.219Benzyl THF/PBS 20.54 0.25 M  6 17 35 0.224 Pyridyl Sulfide THF/PBS 68.580.22 P, MLP*, TP*  7 17 33 0.235 Thiol THF/PBS 95.06 0.55 P, MLP*  8 1730 0.252 Thiol THF/PBS 97.96 0.52 P, MLP*  9 17 23 0.305 Thiol THF/PBS29.78 0.43 M, P, FM 10 45 44 0.38 Benzyl THF/PBS 14.28 0.33 M, FM 11 4538 0.415 Phthalimide THF/PBS 19.03 0.29 M 12 45 24 0.529 Benzyl THF/PBS10.13 0.38 M 13 45 20 0.574 Benzyl THF/PBS 12.75 0.3 M 14 45 12 0.692Benzyl THF/PBS 15.43 0.43 M Com/Aq Solv = Common/Aqueous Solvents usedduring the impingement process. M = Micelles, FM = Filomicelles, BN =Bicontinuous Nanospheres, P = Polymersomes, MLP = MultilamellarPolymersomes, TP = Tubular Polymersomes. Predominant population(s) shownin bold. *Population only found after multiple impingements. **Very rarepopulation. DLS diameter and polydispersity data not available forsamples predominantly composed of filomicelles, i.e. sample 3‡.

We found that multiple impingements through the CIJ mixer both decreasedthe mean polymersome diameter and lowered the PDI to levels achievableby extrusion (FIGS. 2A and 7). To maintain high levels of the commonsolvent within hydrophobic PPS membranes for continued chainflexibility, we impinged a PEG-bl-PPS solution in THF against 1×PBS,collected the resulting mixture without diluting in a reservoir, evenlydivided the THF/polymersome/PBS solution between the CIJ mixer syringesand repeated the impingement. As no reservoir was used, the volumeremained constant, and the impingement could be repeated multiple timesover the course of a several minutes without sample loss. Unexpectedly,the diameter and PDI increased over the first two repeat impingements(2× and 3×), before reducing significantly in the following twoimpingements (4× and 5×) (FIGS. 2A and 7). By the fifth impingement,there was no statistical difference in the diameter or PDI between FNPpolymersomes and polymersomes formed by thin film hydration that weresubsequently extruded through a 0.1 μm filter. Furthermore, 5× impingedpolymersomes remained stable for days and demonstrated no detectablechange in PDI (FIG. 2B). This change in the distribution of sizes can beobserved by cryoTEM from the first impingement to the fifth and finalimpingement (FIGS. 2C-2G). While the first impingement generatedprimarily unilamellar and some rare multilamellar polymersomes rangingbetween 50 nm to nearly a micron in diameter, the 5× impingedpolymersomes were monodisperse (PDI<0.15) and all possessed a singlebilayer (FIG. 2G). Interestingly, the 2× and 3× polymersome populationswere composed of large, multilamellar, and/or tubular polymersomes(FIGS. 2D, 2E, 10C and 10E). Tubular polymersomes have been predicted insimulations of polymersome formation under conditions of shear flow.Multilamellar vesicles may be a result of multiple fusion events inducedby increased polymersome collisions under conditions of turbulent flowwhile PPS chains remain fluid and swollen with THF. Since no changes invesicle polydispersity or structure were observed after 5 impingements,it is possible that continued diffusion of THF out of the PPS domainsreduced chain flexibility sufficiently to prevent polymersome fusion. Insilico simulations of these events will be required to verify ourhypotheses. We found this protocol of repeated impingements to supportthe gram-scale production of monodisperse polymersomes within a matterof minutes, which is a task that could require days to weeks to achieveby alternative fabrication methods such as thin film hydration andextrusion.

PEG-bl-PPS Copolymers Can Form Numerous Nanoarchitectures via FlashNanoprecipitation PEG-bl-PPS can self-assemble into severalnanoarchitectures simply by tuning the PEG weight fraction (f_(PEG)).Using thin film hydration, f_(PEG) between 0.20 and 0.30 formpolymersomes, f_(PEG)˜0.38 forms filomicelles and f_(PEG)>0.40 generallyresults in spherical micelles (FIG. 1B). In order to determine whetherthere is a similar connection between f_(PEG) and morphology whenpolymersomes are formed by FNP, we synthesized the polymers listed inTable 1 and assessed their assembly into nanostructures using cryogenicelectron microscopy (FIG. 3). These polymers were impinged against a 40mM calcein solution in 1×PBS, as calcein is a water-soluble dye and itsencapsulation would confirm the formation of vesicle structures, asopposed to the lipophilic cores of micelles and filomicelles. Thediameters and calcein-loading ability of the assembled nanocarriers areshown in FIG. 3A. Flash nanoprecipitated polymersomes were found tooccupy a space characterized by diameters ranging from 50-200 nm and PEGweight fractions from 0.187-0.305, similar to that for thin filmrehydration (FIG. 3A, dotted oval). Copolymers 10-14 (f_(PEG)>0.40) werefound to form primarily micelles, with a small population offilomicelles detected in the copolymer 10 sample. While copolymer 3assembled polymersomes using PBS as the aqueous nonsolvent, switching topure water resulted in the generation of filomicelles, likely due to theincreased steric stabilization of the PEG corona (FIG. 3G and Table 1).

The three copolymers 1, 4, 5 and 9 (FIG. 3A, arrows), did not follow thepreviously described trends. Copolymer 1 failed to load calcein and wasoutside of the polymersome-forming range, yet had a diameter similar tothat of polymersomes. CryoTEM imaging revealed these nanostructures tobe monodisperse bicontinuous nanospheres (FIG. 3C and FIG. 10D), whichis a nanostructure rarely formed from diblock copolymers in a controlledfashion FNP may therefore present a rapid and scalable alternativemethod of formation of polymeric biocontinuous nanostructures in simpleaqueous solutions without the need for additives or complex blockcopolymer architectures (e.g. dendritic-linear, multiblock and miktoarmstars). Switching the solvent to dimethylformamide (δ=24.8 MPa^(1/2))resulted in the formation of micelles from copolymer 4†, which wasinitially found to assemble vesicles when THF was instead used as thecommon solvent (FIG. 3A, Table 1). This result would be expected due tothe lower similarity in solubility parameters between DMF and PPSrelative to THF and PPS, which would lower chain flexibility and produceslower kinetics for nanostructure transitions. Slowing the transitionkinetics through the use of less similar common solvents is known topromote micelle instead of vesicle formation for other block copolymersystems. DLS and cryoTEM revealed copolymer 5 to form micelles eventhough its f_(PEG) was within the polymersome-forming range (FIG. 8A).This discrepancy can be explained by the higher molecular weight (MW) ofthe PEG block (MW 2000) relative to the other polymersome formingcopolymers (MW 750) (Table 1), as PEG steric stabilization increaseswith MW and strongly impacts nanostructure morphology. Additionally, thelonger PPS chain could result in higher degrees of entanglement andtherefore decreased mobility. Copolymer 9 successfully loaded calcein,but had a diameter closer to that of micelles. CryoTEM found this sampleto be a mix of morphologies, that included a low percentage ofpolymersomes and a dominant micelle population (FIG. 8B).

The alternative vesicular morphologies of multilamellar and tubularpolymersomes (FIGS. 3 and 10A-10A) were detected that were capable ofloading calcein in addition to single-bilayered polymersomes. Themultilamellar polymersomes represented >30% of assembled nanostructuresformed from copolymers 3, 4, 6, 7, and 8 after 2× impingements (FIGS. 3Eand 10E and Table 1). These vesicles were easily distinguishable fromthe relatively common polymersome-within-polymersome structure, as theywere characterized by numerous layers of nested vesicles, often reachinga dozen or more layers deep. Although initially absent, tubular polymerswith lengths commonly exceeding a micron became a dominant population(>50% of assembled aggregates) after 3× impingements of copolymers 3, 4and 6 (FIG. 3B, and Table 1). Tubular polymersome are difficult toassemble either quickly or as the dominant aggregate population, oftenrequiring weeks to form and subsequent separation from more prevalentspherical polymersomes.

Polymersomes Formed via Flash Nanoprecipitation Effectively Load bothHydrophobic and Hydrophilic Molecules A significant benefit ofpolymersomes over micelles and other lipophilic-core nanoparticles isthat their aqueous lumen can encapsulate hydrophilic molecules whilehydrophobic molecules can be simultaneously loaded within the vesiclebilayer. FNP has primarily been limited to the encapsulation ofhydrophobic molecules with a preferred log P greater than 6 for stablenanoparticle formation. Ion-pairing of weakly hydrophobic andhydrophilic payloads with counter-ions to form hydrophobic salts forcomplexation with the block copolymer can allow nanoparticle assembly,but this method is not amenable to the loading of most hydrophilicbiologics. To determine the influence of payload size and watersolubility on loading efficiency into polymersomes, we tested moleculesranging from <1 kDa to >100 kDa and with varying hydrophobicity andStokes radius: calcein, ethyl eosin, indocyanine green, dextran (10 and70 kDa), green fluorescent protein (GFP), and alkaline phosphatase (FIG.4A). With a log P of 1.6, calcein is relatively hydrophilic and small,resulting in the lowest loading efficiency possibly due to its diffusionfrom the interior of assembling polymersomes into the exterior aqueousreservoir before vesicle stabilization. In contrast, both ethyl eosinand indocyanine green presented very high loading efficiencies, as theycan partition into the hydrophobic bilayers during polymersome assembly.The loading efficiency of water soluble macromolecules was significantlyhigher than for calcein and ranged between 15-25% (FIG. 4A). A number ofother variables besides the Stokes radius contribute to the ability fora molecule to permeate through an amphiphilic membrane, includingmolecular shape, hydrophobic affinity, and membrane thickness. All themacromolecules investigated were hydrophilic, and may have had higherloading efficiencies than calcein due to their size; as the largermacromolecules may have become trapped within the nascent polymersomesduring the formation process. This effective encapsulation ofmacromolecules is important for biomedical applications of polymersomes,as many hydrophilic molecules of interest are macromolecules, such asnucleic acids, peptides, and proteins. Co-loading of hydrophilic andhydrophobic molecules into polymersomes was achieved simply bydissolving water soluble molecules in the aqueous stream and lipophilicmolecules in the organic stream prior to impingement within the mixer.The co-loading efficiencies for hydrophilic-hydrophobic pairs ofmolecules are displayed in Table 2. Encapsulation of calcein (490 nmexcitation) and DiI (540 nm excitation) respectively within thepolymersome lumen and membrane produced a FRET emission from DiI whenexposed to a 490 nm light source (FIG. 10A-10E). The ease of thisco-loading process demonstrates that FNP is a powerful tool for theformation of nanocarriers loaded with diverse molecular payloads.

TABLE 2 Loading efficiency for dual-loading by flash nanoprecipitation.All samples fabricated in triplicate. LogP values given when available.Dual Loaded Hydrophilic Hydrophobic Cargoes Loading (%) Loading (%) logPValues TMR-Dextran 70kDa, 16.60 ± 2.98 97.12 ± 7.04  N/A, 9.056 ICGAlkaline Phosphatase, 19.00 ± 5.62  64.91 ± 5.42  N/A, 7.497 Ethyl EosinCalcein, Ethyl Eosin 5.06 ± 1.66 52.02 ± 2.65  1.608, 7.497 Calcein, DiI2.54 ± 2.17 103.47 ± 12.11  1.608, 18.824 GFP, Ethyl Eosin 20.85 ± 6.74 63.71 ± 8.42  N/A, 7.497 Rapamycin, DiD N/A 65.59 ± 7.21  6.181, 19.3887.88 ± 13.11

TABLE 3 Loading efficiency for DNA molecules by flash nanoprecipitation.Polymer Size PDI Zeta potential % loading NH2-PEG1K- 94 ± 1  0.19 ± 0.03 42 ± 3.8 NA PPS82 50 μg DNA 101.8 ± 1    0.21 ± 0.01 30.1 ± 1.2 70loaded 0.5 μg DNA 114.6 ± 2.6  0.18 ± 0.02 39.9 ± 5.3 NA loaded 50 μgDNA in 213.9 ± 7.9  0.43 ± 0.01  0.56 ± 0.26 30 PEK17-PP535

TABLE 4 Positively charged amine-functionalized polymers designed toenhance DNA loading. Polymer Size PDI Zeta potential NH2-PEG1K-PPS82 98± 1 0.17 ± 0.06 44 ± 1 Water NH2-PEG1K-PPS82 94 ± 1 0.19 ± 0.03 42 ± 1PBS

Alkaline Phosphatase remains Enzymatically Active Following Loadingwithin Polymersomes via Flash Nanoprecipitation One concern for the useof FNP with proteins is the mixing of organic solvent with the aqueoussolution, which has the potential to denature the structure of proteinsand decrease their bioactivity. A possible solution to this concern isto load the hydrophilic cargo within the reservoir, after theimpingement has occurred but before the vesicles have fully assembled.To determine the efficiency of this method, we attempted tosimultaneously load polymersomes with two separate fluorescent dextransof the same molecular weight by dissolving one in the impinged aqueousstream and one in the reservoir at the same concentration. The highlywater soluble macromolecule dextran (10 kDa) tagged with either cascadeblue (CB) or fluorescein (F) was used. To avoid any potential influencesof the attached fluorophore on the loading efficiency, we conducted theexperiments in pairs, with the dextran-CB in the syringe and dextran-Fin the reservoir, and vice versa. We found there to be a statisticallysignificant increase in loading via syringe compared to reservoir,though loading by reservoir still occurred at detectable levels (FIG.6A). We further investigated whether loading via reservoir or syringeresulted in a greater amount of functional protein by loading GFP, whichhas a conformation-dependent fluorescence. We found that GFP fluorescedat equal levels whether loaded by syringe or reservoir (FIG. 6B).

The activity of GFP in biologically-relevant systems was furtherexplored by delivering GFP in vitro within polymersomes to bonemarrow-derived dendritic cells (BMDCs). BMDCs were generated from bonemarrow freshly collected from C57BL/6 mice, and after maturation wereplated in the presence of polymersomes. The polymersomes were co-loadedwith the hydrophobic dye ethyl eosin and hydrophilic GFP, and the cellswere further stained with nuclear stain SYTO 61 and lysosome stainLysotracker Blue prior to imaging on a confocal microscope. Punctae ofGFP and ethyl eosin were found within cells, demonstrating thatconformationally-active GFP could be delivered to live cells viapolymersomes (FIG. 11). These results supported previously publishedconfocal microscopy images and verifies that FNP had no impact on theability of PEG-bl-PPS polymersomes to deliver payloads to the cytoplasmof BMDCs.

To further confirm the continued biological activity of loaded proteins,we investigated the function of an enzyme, alkaline phosphatase (AP),encapsulated into polymersomes by FNP. AP removes phosphate groups froma number of substrates, including 5-bromo-4-chloro-3-indolyl-phosphate(BCIP), whose dephosphorylation is detectable by nitro blue tetrazolium(NBT) at an absorbance of 620 nm. We therefore encapsulated AP into thelumen of polymersomes using FNP, and added them to a solution of BCIPand NBT (FIG. 4B). AP-polymersomes were lysed using Triton X-100 toallow the AP, BCIP and NBT to freely react in solution (FIG. 4C). At lowconcentrations of Triton X-100 a low level of background reactivity wasobserved in the system, which was likely due to a burst effect resultingfrom the release of hydrophilic reagents trapped within the vesiclemembranes. The addition of 0.1% Triton X-100 resulted in the continuedformation of significantly more product over a ten-minute period,verifying the retention of AP tertiary conformation and bioactivityfollowing encapsulation within polymersomes by FNP (FIG. 4C).

Low Dosage Subcutaneous Administration of Rapamycin Loaded intoPolymersomes by Flash Nanoprecipitation Reduces Splenic CD4+ and CD8+ TCell Populations in Mice To explore FNP as a method to fabricatepolymersome formulations for the in vivo delivery of therapeutics, weloaded the model immunomodulatory drug rapamycin (Sirolimus), anFDA-approved immunomodulator, into fluorescent polymersomes. Rapamycinhas a low water solubility (log P=6.181) and loaded readily intopolymersomes with an efficiency of 65% (FIG. 4A). Rapamycin inhibits themechanistic target of rapamycin (mTOR) kinase, which is a key regulatorof cell growth, metabolism and proliferation and elicits cellularresponses that are highly dependent on the cell type. Thus, identifyingwhich cells are being influence by rapamycin is critical tounderstanding immune responses generated during its therapeutic use, andthis may be achieved via an immunotheranostic delivery system. In thecase of T cells, mTOR inhibition is known to decrease proliferation,migration and overall population levels for T cells, particularlyCD4+CD25− T cell and effector CD8+ T cell subsets. For dendritic cells,rapamycin has a suppressive effect on maturation and differentiation byinhibiting expression of co-stimulatory molecules and inflammatorycytokines. Macrophages respond to rapamycin by polarizing towards apro-inflammatory M1 phenotype. Although T cells are not phagocytic anddo not associate with polymersomes, their levels and activity can bemodulated via the targeting of antigen presenting cells, such asphagocytic macrophages and dendritic cells. PEG-bl-PPS nanocarriers areendocytosed by phagocytic immune cells in lymph nodes and spleenfollowing SC administration, and we hypothesized that controlleddelivery of rapamycin within polymersomes to these cells may enhanceimmunosuppression at lower dosages than what is typically employedtherapeutically in mice while avoiding uptake of rapamycin by T cells.Rapamycin is administered to mice orally, intraperitoneally, andsubcutaneously, at doses ranging from 75 μg/kg/day to >10 mg/kg/day.Generally, an effective dose for sustained allograft survival in mice isconsidered to be 1.5-3 mg/kg/day.

To demonstrate the difference between rapamycin efficacy in itspolymersome-loaded versus free form, we administered rapamycin to miceonce every three days at an effective average dosage of only 0.33mg/kg/day. After only three administrations of rapamycin loadedpolymersomes, we found that splenic CD4+ and CD8+ T cells weresignificantly reduced in proportion to the total number of T cellscompared to free rapamycin, which instead elicited no significanteffects on T cells (FIGS. 5A, 12A-12H, and flow cytometry gatingstrategy shown in FIG. 13). CD8+ dendritic cells, a dendritic cellpopulation known for superior cross presentation of antigen andresulting CD8+ T cell activation, were also significantly decreased inproportion to the total number of dendritic cells (FIGS. 5A, 12A-12H).The significant drop in CD4+ T cells and CD8+ T cells was accompanied byan increase in the proportion of ‘double negative’ (CD4-CD8-, DN) Tcells (FIG. 5B) and a decrease in the total number of T cells (FIG. 5C).The increase in the proportion of DN T cells was matched by the overalldecrease in T cells, resulting in no significant change in theproportion of DN T cells within the total immune cell population (FIG.14). Although rapamycin has been employed for in vitro and in vivoexpansion of regulatory T cells (Tregs) in mice, we found no significantchanges in Treg levels following controlled delivery withinpolymersomes. Free rapamycin was found to have no significant impact onany immune cell populations at this comparatively low dosage and briefcourse of treatment (FIGS. 5, 12A-12H). The decrease in CD4+ and CD8+ Tcell populations is not likely to have occurred through directintracellular delivery of rapamycin to T cells, as these cells showed noassociation with polymersomes as measured by DiD fluorescence (FIG. 5D).In contrast, phagocytic cells in both the lymph nodes and spleen, suchas macrophages, Ly-6C″ monocytes, and dendritic cells all demonstratedsignificant uptake of polymersomes (FIG. 5D). Modulation of Treg levelsfor T cell suppression in mice via rapamycin has been found to require1.5 mg/kg/day for 14 days or 3 mg/kg/day for 4 days in a separate study,thus it is not surprising that we observed no changes in Treg levels ata dosage of only 0.33 mg/kg/day over the course of 1 week. Thistheranostic strategy thus implicates antigen presenting cells as themediators of our observed rapid low-dosage T cell immunosuppression, anddemonstrates that flash nanoprecipitation can be employed for thefabrication and loading of soft nanoarchitectures capable of controlleddual delivery of imaging agents and therapeutic drugs in vivo.

CONCLUSION

Delivery of drugs in vivo, ranging from hydrophilic to hydrophobic andsmall molecule to protein biologics, remains a significant challenge forthe field of biomedical research. Concerns over efficiency, stability,and scalability have plagued many fabrication processes forself-assembled soft nanoarchitectures, many of which hold vast andunrealized potential for advanced strategies of controlled delivery.Here, we have demonstrated that FNP, a rapid and scalable method ofassembling solid-core block copolymer nanoparticles, can be used toassemble PEG-bl-PPS into monodisperse polymersomes and a variety ofother soft nanoarchitectures, including tubular and multilamellarpolymersomes, filomicelles and rare polymeric bicontinuous nanospheres.FNP is usually performed using block copolymers with high Tg hydrophobicblocks, like polystyrene, that undergo slow structural transitions andallow the formation of stable micellar aggregates. The present resultssuggest that matching the copolymer aggregation rate with the turbulentmixing time during FNP, which can be controlled by the flow rate throughthe mixing chamber, may allow FNP to be applied for the formation ofdiverse metastable nanostructures for other polymers in addition toPEG-bl-PPS. FNP was surprisingly found to be used to effectively load(and co-load) vesicular nanoarchitectures with small molecules andproteins with a wide range of water solubility and molecular weightswhile maintaining their biological activity. The inventors demonstratedthree examples of biological relevance: in vitro delivery of GFP toBMDCs, in vitro enzyme activity of alkaline phosphatase, and in vivodelivery of rapamycin to immune cells, the latter of which demonstratedimmunosuppression of CD4+ and CD8+ T cells at a 5-fold lower dosage thanis typically utilized. These findings significantly expand thecapabilities of FNP and provide new routes for the high throughputnanofabrication of diverse therapeutic nanocarriers.

Materials and Methods

Materials

The following reagents were obtained from Sigma-Aldrich: poly(ethyleneglycol) methyl ether MW 2000 (product 202509), poly(ethylene glycol)methyl ether MW 750 (product 202495), poly(ethylene glycol) MW 300(product 202371), methanesulfonyl chloride (product 471259),triethylamine (product T0886), potassium carbonate (product 791776),thioacetic acid (product T30805), 0.5M sodium methoxide solution(product 403067), propylene sulfide (product P53209), acetic acid(product 695092), benzyl bromide (product B17905),N-(2-bromoethyl)phthalimide (product B66302), 2,2′-dipyridyl disulfide(product 143049), calcein (product C0875), ethyl eosin (product 199540),indocyanine green (product 12633), dichloromethane (product 320269),celite filter cel (product 22139), activated charcoal powder (product161551), anhydrous tetrahydrofuran (product 401757), tetrahydrofuran(product 437638), ethanol (product 459844), methanol (product 179337),diethyl ether (product 346136), deuterated chloroform (product 151823),Tween 80 (product P8074), and sepharose 4B (product 4B200). Thefollowing reagents were purchased from ThermoFisher Scientific:tetramethylrhodamine-dextran 70 kDa (product D1818), fluorescein-dextran10 kDa (product D1820), cascade blue-dextran 10 kDa (product D1976), DiI(product V22885), DiD (product D7757) toluene (product T324), hexanes(product H292), HPLC-grade dimethylformamide (product AA22915K2),Lysotracker Blue DND-22 (product L7525), SYTO 61 (product S11343), ACKlysing buffer (product A1049201), HBSS (product 14175079) and 1×PBStablets (product BP2944). Recombinant A. victoria GFP protein was agenerous gift from the Jewett Lab at Northwestern University. Electronmicroscopy holey carbon 200 mesh copper grids were purchased fromElectron Microscopy Sciences (product HC200CU). Rapamycin was purchasedfrom Selleck Chemicals. Recombinant IL-4, recombinant GM-CSF, and allantibodies for flow cytometry were purchased from BioLegend.

Polymer Synthesis

The synthesis of poly(ethylene glycol)-block-poly(propylene sulfide)(PEG-bl-PPS) was performed as described previously. Briefly, methylether PEG (MW 750 and 2000) were functionalized first with the mesylateleaving group, which was then reacted with thioacetic acid to form aprotected PEG-thioacetate. Base activation of the thioacetate resultedin the formation of a thiolate anion, which was used as the initiatorfor ring opening polymerization of propylene sulfide. The reaction wascompleted with the addition of end capping groups (benzyl bromide orbromoethyl phthalimide), a disulfide-bonding group (dipyridyldisulfide), or the protonation of the thiolate anion with acetic acid,leaving a free thiol at the end of the polymer (PEG-bl-PPS-thiol).Degree of polymerization was assessed via H1 NMR (3H methyl ether, 3.36singlet; 4H PEG —CH₂—CH₂—, wide peak 3.60-3.64; 1H CH₂—CH—CH₃ wide peak2.56-2.65; 2H —CH—CH₂—CH—CH₃, wide peak 2.82-2.95, 3H —CH₂—CH₃ wide peak1.30-1.38).

Nanocarrier Fabrication via Flash Nanoprecipitation

Nanocarriers were formed using the confined impingement jets (CIJ) mixerdescribed by Han et al. PEG-bl-PPS copolymers and hydrophobic moleculesto be loaded within polymersomes were dissolved in 500 μL oftetrahydrofuran (THF) and placed into a 1 mL plastic disposable syringe.500 μL of phosphate buffered saline (1×PBS) and any hydrophilicmolecules to be loaded within the vesicle lumen were prepared in asecond 1 mL syringe. The two solutions were impinged against one anotherwithin the CIJ mixer by hand, at a rate of approximately 1 mL/s. Thesupersaturated solution exited the mixer into a 20 mL glassscintillation vial containing a 2.5 mL reservoir of 1×PBS. This productwas then separated from unloaded molecules and THF on a sepharose 4Bsize exclusion column. For fluorescence measurements, samples were takenboth before and after column purification to assess loading efficiency.

Alternative Nanocarrier Fabrication Techniques

As controls, polymersomes were formed by the standard solvent dispersionand thin film techniques. In the case of solvent dispersion, 20 mg ofpolymer was dissolved in 500 μL of THF, which was dripped into astirring reservoir of 3 mL of 1×PBS. This resulted in an identicalTHF:1×PBS ratio as used for the FNP fabrication method. Hydrophobicmolecules were dissolved in the requisite organic solvent and added tothe polymer/THF solution prior to addition to 1×PBS. Hydrophilicmolecules were first dissolved in the 1×PBS reservoir prior to theaddition of the polymer/THF solution. In the case of thin film, 20 mg ofpolymer was weighed into a 1.8 mL glass HPLC vial and dissolved in 750μL of DCM, which was subsequently removed by vacuum desiccation for 6hours. 1 mL of 1×PBS was then added to the HPLC vial, which was shakenat 1500 rpm overnight on a Multi-Therm shaker (Heidolph) at roomtemperature. Hydrophobic molecules, dissolved in their respectiveorganic solvent, were added to the polymer/DCM solution, and were driedwith the polymer in the vacuum desiccator. Hydrophilic molecules weredissolved first in the 1×PBS prior to its addition to the dried polymerdeposit for hydration under shaking at room temperature.

Co-Loading Experiments

For calcein and ethyl eosin co-loading experiments, the aqueous solventconsisted of 0.4 mM calcein in 1×PBS. 10 μL of a 5 mg/mL ethyl eosinsolution in ethanol was added to 490 μL of THF and 20 mg of polymer. Forthe TMR-dextran and ICG co-loading experiments, 2 mg of TMR-dextran (70kDa) was dissolved in 500 μL 1×PBS. 50 μL of a 1 mg/mL ICG solution inethanol was added to 450 μL of THF and 20 mg of polymer. For alkalinephosphatase-ethyl eosin co-loading experiments, alkaline phosphatase wasloaded at a concentration of 1 mg/mL within the reservoir, while 10 μLof a 5 mg/mL ethyl eosin solution in ethanol was added to 490 μL of THFand 20 mg of polymer. For calcein and DiI co-loading experiments, theaqueous solvent consisted of a 0.4 mM calcein solution in 1×PBS, whichwas impinged against 490 μL THF with 10 μL DiI (as supplied) and 20 mgof polymer. For the experiments examining the relative loading offluorescein-dextran and cascade blue-dextran, both were used from stocksolutions of 1 mg/mL in 1×PBS. For the GFP loading experiments, 50 μL ofa 200 μg/mL was dissolved in 450 μL of 1×PBS (for syringe loading) or2.45 mL 1×PBS (for reservoir loading), while 20 mg of polymer wasdissolved in 500 μL of THF.

Multiple Impingement Experiments

For the multiple impingement samples, the organic and aqueousimpingement solutions were prepared as previously described for thecalcein/ethyl eosin co-loading experiments with a few exceptions. Inthis case, rather than containing 2.5 mL of 1×PBS, the scintillationvial reservoir was initially empty. Following each impingement, theresulting solution containing 1:1 THF:1×PBS, 20 mg of polymer, calcein,and ethyl eosin, was split evenly into two 1 mL syringes, andreintroduced into the CIJ mixer. This process was repeated between oneand four times, with the final impingement emptying into a reservoircontaining 2.5 mL of 1×PBS.

Spectrometric Fluorescence and Absorbance Measurements

Fluorescence and absorbance measurements were taken on a SpectraMax M3microplate reader (Molecular Devices). All readings were taken inblack-walled clear bottom 96-well plates (Corning 07-200-567), at 100 μLvolumes. All readings were normalized to a 100 μL 1×PBS blank control.The following settings were used for fluorescence measurements for eachfluorophore (excitation/emission, filter used): calcein: 470/509, 495filter), ethyl eosin: 525/560, 550 filter, tetramethylrhodamine:555/580, 570 filter, fluorescein: 494/524, 515 filter, cascade blue:400/420, 420 filter, indocyanine green: 780/820, no filter, DiI:549/565, 550 filter, DiD: 644/670, 665 filter and GFP: 485/535, 495filter. NBT diformazan was detected via absorbance at 620 nm.

Rapamycin Loading Efficiency by HPLC UV Absorbance

50 μL aliquots of formulations containing rapamycin were frozen at −80 Cfor at least 3 hours, then were lyophilized overnight. Lyophilizedsamples were dissolved in HPLC grade DMF and were vortexed then brieflycentrifuged to pellet salt from the formulation. Samples were run on aThermo Fisher Dionex UltiMate 3000 system with an Agilent Polypore7.5×300 mm column with an Agilent Polypore 7.5×50 mm guard column,housed at 60 C. The organic mobile phase was HPLC grade DMF, run at 0.5mL/min. Rapamycin was detected using UV absorbance at 270 nm. Forloading efficiency, aliquots were taken from samples before and aftercolumn purification. Analysis was performed using the Thermo ScientificChromeleon software.

Alkaline Phosphatase Enzyme Activity Assay

Alkaline phosphatase was dissolved at 1 mg/mL in 2.5 mL of 137 mM NaClsolution for the reservoir. Unloaded polymersomes were formed bysimilarly but without alkaline phosphatase. All polymersome samples werepurified by size exclusion chromatography (sepharose 4B) to remove THFand unloaded cargo. A stock solution of premixed NBT and BCIP was used(560 μM BCIP and 480 μM NBT prepared in 10 mM Tris and 59.3 mM MgCl₂).40 μL of polymersomes was added to wells of a 96 well plate, along with10 uL of NBT/BCIP solution. Cargo release was performed by the additionof 50 μL of Triton X-100 solution, with final concentrations of TritonX-100 of 0.1%, 0.05%, and 0.01%. Absorbance measurements were takenevery two minutes for 10 minutes.

Size Measurements

Hydrodynamic diameters of nanostructures were measured usingnanoparticle tracking analysis (NTA) on a Nanosight NS300 (Malvern).Measurements were taken using samples at a 1:1000 dilution in 1×PBS,resulting in approximately 0.1 mg/mL polymer concentrations. Readingswere performed using a 633 nm laser. Five 1 minute videos were recordedper sample, with results averaged across the five readings. For micellarsamples, typically >20 nm in diameter, dynamic light scatteringmeasurements were performed using a Zetasizer (Malvern) to moreaccurately measure the size distribution, due to the limitations of NTAfor smaller diameter aggregates.

Log P Values

Log P values were pulled from the ZINC15 database (zinc15.docking.org).

Cryogenic Transmission Electron Microscopy

Specimens for cryoTEM were prepared by applying 4 μL of 1 mg/ml sampleon a pretreated, holey carbon 400 mesh TEM grids and were plunge-frozenwith a Gatan Cryoplunge freezer. Images were collected in vitreous iceusing a JEOL 3200FSC transmission electron microscope operating at 300keV at 4,000×nominal magnification. A total dose of ˜10 e⁻/Å² and anominal defocus range of 2.0-5.0 μm were used. Micrographs were acquiredusing a Gatan 3.710×3,838 pixel K2 Summit direct electron detectoroperating in counting mode. Each micrograph was acquired as 20-framemovies during a 5 s exposure. After data acquisition, the individualframes of each micrograph were aligned using Digital Micrograph software(Gatan) to compensate for stage and beam-induced drift, and the alignedimages were summed for further image processing.

Animals

C57BL/6 male mice, 6-8 weeks old, were purchased from Jackson Labs. Allmice were housed and maintained in the Center for Comparative Medicineat Northwestern University. All animal experimental procedures wereperformed according to protocols approved by the Northwestern UniversityInstitutional Animal Care and Use Committee (IACUC).

Live-Cell Confocal Microscopy

Bone marrow derived dendritic cells were generated from bone marrowcollected from the tibias and femurs of C57BL/6 mice, in a protocolslightly modified from those described previously. Tibias and femurs ofC57BL/6 mice were cleaned of tissue, cut, and flushed through with 10%FBS in HBSS. Cells were centrifuged, supernatant was removed, and theywere then treated with ACK lysis buffer for 5 minutes. Lysis was stoppedwith the addition of excess HBSS, and cells were centrifuged again,resuspended in 10 mL 10% FBS 1× Penstrep RPMI, and were plated in 100 mmpetri dishes. Every three days, 200 ng of GM-CSF and 100 ng of IL-4 wereadded to culture. On the 8^(th) day of maturation, cells were collectedand plated into a FluoroDish at 3×10⁵ cells/mL, along with 15 uL ofdual-loaded GFP ethyl eosin polymersomes. Lysotracker Blue (lysosomestain) and SYTO 61 (nuclear stain) were added at 1:10,000 dilutions.Plated cells were imaged within a humidified chamber using a 63×oil-immersion objective on a SP5 Leica Confocal Microscope using HyDdetectors and four lasers: 415 nm diode laser for Lysotracker Blue, 488nm argon laser for GFP, 514 nm argon laser for ethyl eosin, and 633 nmHeNe laser for SYTO 61.

In Vivo Rapamycin Delivery

Formulations of rapamycin polymersomes and blank polymersomes wereformed by flash nanoprecipitation using 20 mg of PEG17-bl-PPS36-Thiolpolymer, with or without 0.5 mg rapamycin, respectively, dissolved inTHF. Sterile 1×PBS was used as the aqueous phase and reservoir solution.Fluorescently labeled polymersomes of both formulations were formedsimilarly, with the addition of 25 μg of DiD in the organic phase. Aformulation of free rapamycin was made in a solution of 8% ethanol, 10%PEG300, and 10% Tween 80 in 1×PBS. Briefly, rapamycin was dissolved inethanol (3 mg/mL), and 31.2 μL was added to 1 mL of a solution of 10%PEG300 and 10% Tween 80 in 1×PBS (3.1% ethanol final, ˜125 μg/mLrapamycin concentration). Vehicle was also injected without rapamycin,in which case 31.2 μL of pure ethanol was added to the 10% PEG300 10%Tween 80 solution. Mice were injected subcutaneously, slightly anteriorto the scapula, with 1 mg/kg doses of rapamycin, or equivalentinjections of vehicle or blank polymersomes, N=3 per treatment group.Injections were performed on days 1, 4 and 7, with the final set ofinjections containing fluorescently labeled polymersomes, whenapplicable. Mice were sacked on day 8, and the draining (brachial) lymphnodes were collected, along with the spleens. Organs were mechanicallyhomogenized in RPMI media and passed through a 70 μm cell strainerbefore being stained for flow cytometry.

Flow Cytometry

Splenic cells were first treated with ACK lysis buffer for 5 minutes onice before being spun down and resuspended in blocking buffer. Cellswere stained with Zombie Aqua as a fixable live/dead stain and FcRs wereblocked with anti-mouse CD16/CD32. Cells were stained with a cocktail ofantibodies in three panels. Panel 1: anti-mouse CD45 FITC, anti-mouseCD3 APC-Cy7, anti-mouse CD4 PE-Cy5, anti-mouse CD8a PE-Cy7, anti-mouseCD19 Pacific Blue, anti-mouse CD49b PerCp-Cy5.5, and anti-mouse CD25 PE.Panel 2: anti-mouse CD11b PerCp-Cy5.5, anti-mouse CD11c Pacific Blue,anti-mouse I-A/I-E FITC, anti-mouse B220 PE, anti-mouse Gr-1 APC-Cy7,and anti-mouse CD8a PE-Cy7. Panel 3: anti-mouse CD11b PerCp-Cy5.5,anti-mouse CD11c Pacific Blue, anti-mouse F4/80 FITC, anti-mouse Ly-6CAPC-Cy7, and anti-mouse Ly-6G PE-Cy7. After washes, cells were fixed byIC cell fixation buffer (Biosciences). Flow cytometry was performed withFACSDiva on a LSRII flow cytometer (BD Biosciences), with the APCchannel used to detect the DiD loaded into polymersomes. Data wasanalyzed using CytoBank online software.

Statistical Analysis

Statistical significance for changes in nanostructure diameter andpolydispersity by multiple impingements and changes in loadingefficiency by loading method were determined using Student's t-test,with multiple comparison correction via the Holm-Sidak method,alpha=0.05. All other statistical significance was determined by 2-wayANOVA and Tukey's multiple comparison test, alpha=0.05. In all graphs,*=p<0.05, **=p<0.01, ***=p<0.001, and ****=p<0.0001. All statisticalanalysis was performed using GraphPad Prism.

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We claim:
 1. A bicontinuous nanosphere nanocarrier comprisingpoly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS).
 2. Thenanocarrier of claim 1, wherein the bicontinuous nanosphere comprisesboth aqueous and lipophilic cores.
 3. The nanocarrier of claim 1,wherein the PEG-bl-PPS has a glass transition temperature between about−40° C. and about 0° C.
 4. The nanocarrier of claim 1, wherein thePEG-bl-PPS is PEG₁₇-bl-PPS₃₀-Thiol.
 5. The nanocarrier of claim 1,wherein the nanocarrier additionally comprise one more target molecules.6. The nanocarrier of claim 5, wherein the target molecule is selectedfrom the group consisting of a DNA molecule, an RNA molecule, a plasmid,a peptide, a protein, and combinations thereof.
 7. The nanocarrier ofclaim 5, wherein the nanocarrier comprises a hydrophobic target moleculeand a hydrophilic target molecule.
 8. The nanocarrier of claim 1,wherein the nanocarrier is less than 1060 nm in diameter.
 9. Thenanocarrier of claim 8, wherein the nanocarrier is less than 700 nm indiameter.
 10. A composition comprising the nanocarrier of claim 1 and apharmaceutically acceptable carrier.
 11. A bicontinuous nanospherenanocarrier produced by a flash precipitation method comprising thesteps of: (i) providing an organic phase solution comprisingpoly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) and aprocess solvent, wherein the PEG-bl-PPS has a glass transitiontemperature below 0° C., (ii) providing an aqueous phase solutioncomprising an aqueous solvent, (iii) mixing the organic phase solutionand the aqueous phase solution by impingement to form a mixture, and(iv) introducing the mixture into a reservoir to cause precipitation ofthe PEG-bl-PPS as a bicontinuous nanosphere.
 12. The nanocarrier ofclaim 11, wherein the organic phase and the aqueous phase are impingedonce.
 13. The nanocarrier of claim 11, wherein PEG weight fraction ofthe PEG-bl-PPS is about 0.12.
 14. The nanocarrier of claim 13, whereinthe PEG-bl-PPS is PEG₁₇-bl-PPS₃₀-Thiol.
 15. The nanocarrier of claim 11,wherein the bicontinuous nanosphere is monodisperse.
 16. The nanocarrierof claim 11, wherein the process solvent is selected from the groupconsisting of tetrahydrofuran (THF), dimethylformamide (DMF), anddimethyl sulfoxide (DMSO).
 17. The nanocarrier of claim 11, wherein theaqueous solvent is water.
 18. The nanocarrier of claim 11, additionallycomprising a target molecule.
 19. The nanocarrier of claim 18, whereinthe organic phase solution additionally comprises a hydrophobic targetmolecule.
 20. The nanocarrier of claim 18, wherein the aqueous phasesolution additionally comprises a hydrophilic target molecule.